A replication clock for Mycobacterium tuberculosis

Abstract

Few tools exist to assess replication of chronic pathogens during infection. This has been a considerable barrier to understanding latent tuberculosis, and efforts to develop new therapies generally assume that the bacteria are very slowly replicating or nonreplicating during latency [1][2][3] . To monitor Mycobacterium tuberculosis replication within hosts, we exploit an unstable plasmid that is lost at a steady, quantifiable rate from dividing cells in the absence of antibiotic selection. By applying a mathematical model, we calculate bacterial growth and death rates during infection of mice. We show that during chronic infection the cumulative bacterial burden-enumerating total live, dead and removed organisms encountered by the mouse lung-is substantially higher than estimates from colony forming units. Our data show that M. tuberculosis replicates throughout the course of chronic infection of mice and is restrained by the host immune system. This approach may also shed light on the replication dynamics of other chronic pathogens.Many deadly diseases, such as AIDS, malaria and tuberculosis, result from infections that persist for long periods. Disease processes that unfold over months or years are especially difficult to model in the laboratory. New approaches are sorely needed to generate insight into the pathogenesis and treatment of such agents. 8,[11][12][13] . Additionally, isoniazid, the first-line agent used to treat latent tuberculosis, has virtually no efficacy against nonreplicating bacilli in vitro [14][15][16] or in vivo 17 . Similarly, the rapid reactivation of latent Mtb after anti-tumor necrosis factor-α therapy 18 seems inconsistent with infection by bacteria in a nonreplicating state.To reassess mycobacterial replication, we developed a method based on pBP10, a circular plasmid carrying a kanamycin resistance marker 19 . Plasmids replicate coordinately with bacterial DNA, sometimes requiring antibiotic selection for their maintenance. Without selection, plasmids are lost from a proportion of daughter cells during cell division, and thus decreasing plasmid numbers can act as a marker of division. We introduced pBP10 into Mycobacterium smegmatis and Mtb to evaluate the feasibility of using this plasmid as a replication clock.M. smegmatis-pBP10 was maintained in log-phase culture under conditions where doubling times ranged from 2 h to 5 h. Plasmid loss was determined by plating on agar with and without kanamycin. The plasmid was steadily lost as cells divided, with a rate proportional to the growth rate under each condition (Fig. 1a,b). The plasmid loss per generation was constant, regardless of the replication rate (Fig. 1c). We applied a mathematical model (Supplementary Methods online) to determine the segregation constant s -the frequency of daughter cells losing plasmid per generation. This value (0.11 ± 0.0074 (s.d.)) was reproducible across all tested growth conditions.Mtb-pBP10 was maintained in log phase for ~30 generations by subculturing every 2-3 d without antibiotics unde...

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

“…174 In the standard antituberculosis regimen, rifampicin and isoniazid both have a short half-life of 2-3 h and pyrazinamide has a half-life of 10 h, while M tuberculosis has a doubling time of 14-96 h and the mutation rates (2·56 × 10 − ⁸ for isoniazid, 2·56 × 10 − ⁷ for ethambutol, and 2·25 × 10 − ¹⁰ for rifampin) identified by David, 167 with a The Lancet Respiratory Medicine Commission total bacterial burden of 10⁸ in a cavity. [175][176][177][178] The antibiotics are no longer present because clearance occurs before a single M tuberculosis has replicated, and certainly by the second and third replications. This timing makes the probability of generation of mutants, or even amplifying pre-existing ones, less likely, particularly for nonreplicating persistors and semi dormant bacilli, aptly described as "fat and lazy" by Garton and colleagues 179 because of their lipid content and slow doubling times, which can take weeks.…”

Insights on the paleoepidemiology of ancient tuberculosis from the structural analysis of postcranial remains from the Ligurian Neolithic (northwestern Italy)

“…Changes in the pathogen as well as host immunity may significantly alter TB morbidity and virulence over a relatively short period of time (Palkovich, 1981). In particular, murine models suggest that the chronic slow progression of TB is not due to slow replication of the bacterium itself, but to the ability of the localized immune response to maintain equilibrium and latency (Munoz-Elias et al, 2005;Ulrichs et al, 2005;Gill et al, 2009). There is also clinical evidence suggesting that the host response plays a major role in restricting intracellular mycobacterial growth and therefore in determining the clinical manifestations of the disease (Schluger and Rom, 1998).…”

Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice

“…In the i.v. infection model, bacteria replicate exponentially in the lungs for the first 2 wk postinfection; thereafter, bacterial numbers remain relatively stable for several months (6)(7)(8). From 4 wk onward, half of the mice were treated with INH.…”

“…Although these drugs rapidly kill Mycobacterium tuberculosis grown in vitro, they are less active against bacteria grown in vivo in mammalian hosts (4,5). In the mouse model of TB, infection progresses through a brief acute phase, when bacteria replicate exponentially in the lungs, to a protracted chronic phase, when growth slows and bacterial numbers are stabilized by the immune response (6)(7)(8). Anti-TB drugs like INH display good bactericidal activity in acutely infected mice, but chronic infection is more refractory (9).…”